Assessing cell proliferation dynamics is crucial to understand the spatiotemporal control of organogenesis. Here we have generated a versatile fluorescent sensor, PlaCCI (plant cell cycle indicator) on the basis of the expression of CDT1a-CFP, H3.1-mCherry and CYCB1;1-YFP, that identifies cell cycle phases in Arabidopsis thaliana. This tool works in a variety of organs, and all markers and the antibiotic resistance are expressed from a single cassette, facilitating the selection in mutant backgrounds. We also show the robustness of PlaCCI line in live-imaging experiments to follow and quantify cell cycle phase progression.Development in multicellular organisms depends on the highly regulated formation of new organs. Organogenesis requires the production of a sufficient number of cells in a temporally and spatially regulated manner, that eventually will constitute the cohort of different cell types present in the organ. Therefore, understanding morphogenesis requires the availability of confident markers to assess cell proliferation and identify unequivocally the different cell cycle phases in a living cell environment.Efforts in several animal models have led to the development of the FUCCI (fluorescent, ubiquitination-based cell cycle indicator) systems that identify each cell cycle phase [1][2][3][4] . Comparable advances for live-imaging of cell proliferation studies in plants lag behind. One reason is that plants lack the CDT1a-interacting protein, geminin, a key component of the FUCCI system, although they contain a CDT1a-interacting protein (GEM) that is structurally unrelated to animal geminin 5,6 . Attempts to circumvent this have been reported. Thus, in addition to the tags used for G2 + M (refs. 7,8 ), the expression of a truncated version of CDT1a, a DNA replication initiator protein, from a heterologous promoter has been reported as an S + G2 marker 9 . In another approach, the fluorescent protein Venus fused to the destruction box of CYCB1;1 was expressed under the control of H4 promoter and labelled cells in S + G2 + M (ref. 10 ). However, a comprehensive cell cycle marker line should ideally (1) be able to distinguish in an unequivocal manner all cell cycle phases to follow individual cells in a growing organ and (2) offer the possibility of using advanced live-imaging strategies. This is precluded when using nucleoside analogues for labelling the S-phase, since they require specific detection protocols incompatible with live imaging.Still, one major drawback of cell cycle marker lines developed so far is the lack of appropriate markers to identify unequivocally cells in G1. We have recently analysed in detail CDT1a dynamics in proliferating cells and demonstrated that CDT1a starts to be loaded into chromatin shortly after mitosis, reaches a maximum during G1 and is rapidly degraded only minutes after S-phase initiation 11 , expanding previous claims of its proteasome-dependent degradation 12 . Importantly, we demonstrated that CDT1a accumulates rapidly after the S-phase of endocycling cells and is...

Purine analogs are among the most effective chemotherapeutic drugs for the treatment of chronic lymphocytic leukemia (CLL). However, chemoresistance and toxicity limit their clinical use. Here, we report that the DNA polymerase inhibitor aphidicolin, which displayed negligible cytotoxicity as a single agent in primary CLL cells, markedly synergizes with fludarabine and cladribine via enhanced apoptosis. Importantly, synergy was recorded regardless of CLL prognostic markers. At the molecular level, aphidicolin enhanced purine analog-induced phosphorylation of p53 and accumulation of γH2AX, consistent with increase in DNA damage. In addition, aphidicolin delayed γH2AX disappearance that arises after removal of purine analogs, suggesting that aphidicolin causes an increase in DNA damage by impeding DNA damage repair. Similarly, aphidicolin inhibited UV-induced DNA repair known to occur primarily through the nucleotide excision repair (NER) pathway. Finally, we showed that fludarabine induced nuclear import of XPA, an indispensable factor for NER, and that XPA silencing sensitized cell lines to undergo apoptosis in response to fludarabine. Together, our data indicate that aphidicolin potentiates the cytotoxicity of purine analogs by inhibiting a DNA repair pathway that involves DNA polymerases, most likely NER, and provide a rationale for manipulating it to therapeutic advantage.

Breast cancer is the most common malignancy among women worldwide. Over the last four decades, diagnostic and therapeutic procedures have improved substantially, giving patients with localized disease a better chance of cure, and those with more advanced cancer, longer periods of disease control and survival. However, understanding and managing heterogeneity in the clinical response exhibited by patients remains a challenge. For some treatments, biomarkers are available to inform therapeutic options, assess pathological response and predict clinical outcomes. Nevertheless, some measurements are not employed universally and lack sensitivity and specificity, which might be influenced by tissue-specific alterations associated with aging and lifestyle. The first part of this article summarizes available and emerging biomarkers for clinical use, such as measurements that can be made in tumor biopsies or blood samples, including so-called liquid biopsies. The second part of this article outlines underappreciated factors that could influence the interpretation of these clinical measurements and affect treatment outcomes. For example, it has been shown that both adiposity and physical activity can modify the characteristics of tumors and surrounding tissues. In addition, evidence shows that inflammaging and immunosenescence interact with treatment and clinical outcomes and could be considered prognostic and predictive factors independently. In summary, changes to blood and tissues that reflect aging and patient characteristics, including lifestyle, are not commonly considered clinically or in research, either for practical reasons or because the supporting evidence base is developing. Thus, an aim of this article is to encourage an integrative phenomic approach in oncology research and clinical management.